Use of apoptosis inhibiting compounds in degenerative neurological disorders

ABSTRACT

The invention provides methods and compositions for localized delivery of a vector comprising a therapeutic agent to a specific region of the brain associated with a neurodegenerative diseases that is characterized by an excess buildup of buildup of intracellular protein aggregates. In particular, the invention provides methods and compositions used to deliver an adeno-associated virus vector (AAV) comprising a nucleotide sequence encoding an inhibitor of apoptosis protein (IAP) to cells in the region.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/686,588 filed on Jun. 2, 2005 and entitled “Use of ApotosisInhibiting Compounds in Degenerative Neurological Disorders,” and toU.S. Provisional Patent Application No. 60/621,307 filed on Oct. 22,2004 and entitled “Use of 1AP-Proteosome Inhibiter in DegenerativeNeurological Disorders.”

BACKGROUND OF THE INVENTION

The invention is generally in the field of methods and compositions fortreating neurodegenerative diseases characterized by excess buildup ofintracellular protein aggregates such as Parkinson's disease (PD), usingviral and non-viral delivery systems that deliver therapeutic agents tospecific regions of the brain. More specifically, using anadeno-associated viral vector to deliver a nucleotide sequence encodingan inhibitor of apoptosis protein (IAP) to specific regions of the brainassociated with such neurodegenerative diseases.

Neurodegenerative diseases are generally characterized by a degenerationof neurons in either the brain or the nervous system of an individual.Neuronal cell death can occur as a result of a variety of conditionsincluding traumatic injury, ischemia, degenerative disease (e.g.,Parkinson's disease, ALS, or SMA), or as a normal part of tissuedevelopment and maintenance. In addition to Parkinson's disease, variousother diseases, such as Huntington's disease, Alzheimer's disease andMultiple Sclerosis, ALS, fall within this category. These diseases aredebilitating and the damage that they cause is often irreversible.Moreover, in the case of a number of these diseases, the outcome isinvariably fatal.

Developmental cell death, or apoptosis has been implicated inneurodegenerative diseases. Apoptosis is a naturally occurring processthought to play a critical role in establishing appropriate neuronalconnections in the developing central nervous system (CNS). Apoptosis ischaracterized morphologically by condensation of the chromatin followedby shrinkage of the cell body. Biochemically, the hallmark of apoptosisis the degradation of nuclear DNA into oligonucleosomal fragments. DNAladdering precedes cell death and may be a key event leading to death.

Progress is being made on many fronts to find agents that can arrest theprogress of these diseases. Nonetheless, the present therapies for most,if not all, of these diseases provide very little relief. One problemhas been the relevance of current animal models to human disease. Todate, the cause of neuronal death has remained elusive. Thegold-standard animal models for PD involve rapid destruction of dopamineneurons using chemicals which are fairly specific for dopamine neurons.These chemical toxins, which include 6-hydroxydopamine (6OHDA) and MPTP,cause oxidative damage to dopamine neurons in both rodents and primates.These models can be useful to test the efficacy of new therapiesdesigned to improve the symptoms of PD, since such treatments aredesigned to intervene after cells have died or become dysfunctional,regardless of the cause of cell death. In order to test the value ofprotective or curative strategies, however, the mechanism of cell deathmust be relevant to human disease otherwise successful experimentalstudies will not translate into effective human therapy.

Accordingly, a need exists to develop therapies that can alter thecourse of neurodegenerative diseases. More generally, a need exists forbetter methods and compositions for the treatment of neurodegenerativediseases in order to improve the quality of the lives of those afflictedby such diseases.

SUMMARY OF INVENTION

The invention is based, at least in part, on the discovery thatlocalized delivery of a vector comprising an apoptosis inhibiting agentto a specific region of the brain associated with a neurodegenerativediseases characterized by excess buildup of intracellular proteinaggregates, can promote the improvement of the neurodegenerativedisease. The apoptosis inhibiting agent can either be an inhibitor ofapoptosis protein, or a nucleic acid molecule that inhibits expressionof a target protein involved in apoptosis, such as RNA (e.g., RNAinterference). In particular, the invention pertains to methods andcompositions used to deliver a vector, (e.g., an adeno-associated virusvector (AAV)) comprising a nucleotide sequence encoding an inhibitor ofapoptosis protein (IAP) e.g., X-linked inhibitor of apoptosis protein(XIAP) to target cells, (e.g., the substantia nigra pars compact).

It appears that abnormal proteasome activity in neuronal cells is acontributing factor in neurodegenerative diseases such that the cellslose their ability to adequately degrade proteins, especially themutated or misfolded proteins that may be pathological components ofneurodegenerative diseases. Insofar as loss of function, or change infunction, of the proteasome is a contributing factor in neurondegeneration. It has been discovered that blocking apoptotic cell deathprotects neurons from death following proteasome inhibition in vivo. Theinvention provides a method for inhibiting death of a cell of thenervous system, e.g., a neuron. Compositions comprising an inhibitor ofapoptosis, e.g., an X-linked inhibitor of apoptosis protein (XIAP),which act as a potent inhibitor of caspases, e.g., caspases 9, 3 and 7,provide a therapeutic neuroprotective effect. The methods includeincreasing the biological activity (e.g., levels or neuroprotectiveeffects) of a inhibitor of apoptosis protein in a region of the centralnervous system.

Accordingly, in one aspect, the invention pertains to a method fortreating a neurodegenerative disease in a subject by identifying atarget site in the central nervous system that requires modification,and delivering a vector comprising a nucleotide sequence encoding aninhibitor of apoptosis protein (IAP), or a fragment thereof, to thetarget site in the central nervous system. The fragment encodes apeptide. The IAP is expressed in the target site to treat or reduce theneurodegenerative disease. The neurodegenerative disease is preferablywith a neurodegenerative diseases characterized by excess buildup ofintracellular protein aggregates. Examples of such neurodegenerativedisease include, but are not limited to, Parkinson's disease,Huntington's disease, Alzheimer's disease, senile dementia, and AmyloidLateral Schlerosis (ALS).

In particular, the invention pertains to methods and compositions usedto deliver a vector, (e.g., an adeno-associated virus vector (AAV))comprising a nucleotide sequence encoding inhibitor of apoptosis protein(IAP), or a peptide fragment thereof, to target cells, e.g., substantianigra pars compacta. Examples of apoptosis protein (IAP) includes, butis not limited to, X-linked inhibitor of apoptosis protein (XIAP), NIAP,cIAP-1 and cIAP-2, and is preferably, XIAP. In a preferred embodiment,the inhibitor of apoptosis protein is a peptide fragment of XIAP, suchas a peptide fragment comprising the BIR-3 domain.

Particularly preferred methods of delivering the vector to specificregions of the brain are those techniques that are simple, safe, andhave a lower risk associated with them than lesioning, electrodeimplantation or cell transplantation. For example, delivery of thevector using stereotactic microinjection techniques, or delivery of thevector using specialized probes, or percutaneous delivery via disruptionof the blood-brain barrier. Delivery of the vector using the method ofthe invention results in minimal immunological or inflammatory responseswithin the regions of the brain, thus eliminating the need forimmunosupression. After delivery of the vector to a specific region ofthe brain, regional dispersion and/or diffusion of vector occursensuring local distribution of gene and stable gene expression.

Suitable vectors for delivery include viral vectors and non-viraldelivery methods such as liposome-mediated delivery vector. Examples ofviral vectors include, but are not limited to, adeno-associated viralvector adenovirus vectors, herpes virus vectors, parvovirus vectors, andlentivirus vectors, preferably, adeno-associated viral vector.

The regions of the brain that can be targeted are any regions typicallyassociated with neurodegenerative diseases, and which can be targetedusing standard procedures such as stereotaxic delivery. Examples ofbrain regions include, but are not limited to, the basal ganglia,subthalmic nucleus (STN), pedunculopontine nucleus (PPN), substantianigra (SN), thalmus, hippocampus, the substantia nigra pars compacta,cortex, and combinations thereof.

In another aspect, the invention pertains to a method for treatingParkinson's disease in a subject by identifying one or more regions ofthe brain that require modification, and delivering a vector comprisinga nucleotide sequence encoding an inhibitor of apoptosis protein (IAP)to the region of the brain. The IAP is expressed in the region of thebrain to treat or reduce Parkinson's disease.

In another aspect, the invention pertains to a method for treatingHuntington's disease in a subject by identifying one or more regions ofthe brain that require modification, and delivering a vector comprisinga nucleotide sequence encoding an inhibitor of apoptosis protein (IAP)to the region of the brain. The IAP is expressed in the region of thebrain to treat or reduce Huntington's disease.

In yet another aspect, the invention features a vector for expression ofan IAP in cells of the central nervous system comprising a tissuespecific promoter operably linked to a nucleic acid encoding an IAP, anda post-transcriptional regulatory element.

In one embodiment, the promoter is specific for central nervous systemcells and tissues, such as the cells and tissues of the brain. In apreferred embodiment, the promoter is the CMV/CBA promoter. The vectoralso preferably comprises post-transcriptional regulatory elements toenhance expression of the encoded protein. In a preferred embodiment,the post-transcriptional regulatory element is the woodchuckpost-transcriptional regulatory element.

In another aspect, the invention pertains to a chimeric peptide formodulating a neurodegenerative disease comprising a peptide fragment ofan inhibitor of apoptosis protein (IAP) operably linked to a signalpeptide. The chimeric peptide may further comprise a TAT domain. Theinhibitor of apoptosis protein (IAP) can be X-linked inhibitor ofapoptosis protein (XIAP) or a fragment thereof. The peptide fragment cancomprise the BIR3 domain of XIAP. The peptide fragment can also be anXIAP fragment comprising a mutation. The mutation may occur in the oraround the BIR3 domain of XIAP. Mutant fragments can comprise at leastone mutation at an amino acid position selected from the groupconsisting of D148, H343, D214, E314, and W310, or combinations thereof.In one embodiment, least one mutation results in the chimeric peptidehaving neuroprotective properties. In another embodiment, least onemutation results in a chimeric peptide that blocks Smac activity.

In yet another aspect, the invention pertains to a chimeric peptide formodulating a neurodegenerative disease comprising a BIR3 peptidefragment of X-linked inhibitor of apoptosis protein (XIAP) operablylinked to a signal peptide and a TAT domain. The BIR3 peptide fragmentcomprises at least one mutation at an amino acid position selected fromthe group consisting of D148, H343, D214, E314, and W310, orcombinations thereof. At least one of these mutations results in achimeric peptide having neuroprotective properties and/or one thatblocks Smac activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the rAAV vectors of theapplication

FIG. 2A illustrates the reduction of cell death that occurs in cellstransfected with XIAP plasmids.

FIG. 2B is a schematic representation of the pCAspace3-Sensor vector.

FIG. 2C illustrates the location of apoptotic proteins through anapoptosis assay using the vector represented in FIG. 2B.

FIG. 2D illustrates the results of an assay to determine the effect ofXIAP and dXIAP on caspase-3 inactivation.

FIGS. 3A-3D show the effect of XIAP administration in in vitro models offamilial Parkinson's and Huntington's disease.

FIGS. 4A-4D illustrate the effects of rAAV-mediated dXIAP delivery oncell survival of cells treated with proteosome inhibitors.

FIGS. 5A-5G illustrate XIAP's protective effect on neurons of thesusbtantia nigra in a PSI (rat) model of Parkinson's disease.

FIG. 6A is the known amino acid sequence of human XIAP including theBIR1, Linker, BIR2, BIR3 and RING domains.

FIG. 6B is the amino acid sequence of the BIR3 domain of human XIAP.

FIG. 6C shows locations of the various mutations performed on XIAP.

FIG. 7 illustrates the effects of XIAP point mutants onpolyglutamine-induced cell death.

FIG. 8 illustrates the effects of XIAP point mutants on PSI-induced celldeath.

FIG. 9 illustrates the effects caspase inhibition on polyglutamineinduced cell death.

FIG. 10 illustrates the effects caspase inhibition on PSI induced celldeath.

DETAILED DESCRIPTION

The practice of the present invention employs, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. (See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, Vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, Vol. I & II (P. Tijessen, ed.);Fundamental Virology, 2nd Edition, Vol. I & II (B. N. Fields and D. M.Knipe, eds.)).

So that the invention is more clearly understood, the following termsare defined:

The phrase “apoptosis inhibiting agent” as used herein refers to amolecule that is an inhibitor of apoptosis protein, or a nucleic acidmolecule that inhibits expression of a target protein involved inapoptosis, such as RNA (e.g., RNA interference). Examples of apoptosisinhibiting agents include but are not limited to a class of compoundsfrom the BIR family, e.g. SURVIVIN and BRUCE.

The term “apoptosis” as used herein refers to the art recognized use ofthe term for an active process of programmed cell death characterized bymorphological changes in the cell. Apoptosis is characterized bymembrane blebbing and nuclear DNA fragmentation. Apoptosis can occur viatwo pathways, the caspase-dependent pathway, which involves caspases, aninhibitor of apoptosis protein (IAP) and activation of the caspasepathway. Alternatively, apoptosis can occur via the caspase-independentpathway, which does not involve caspases.

The term “zymogen” as used herein refers to the inactive proform of anenzyme e.g. a caspase, which is typically activated by proteolysis.

The term “caspase” as used herein refers to a cysteine protease thatspecifically cleaves proteins after Asp residues. Caspases exist asinactive proenzymes which undergo proteolytic processing at conservedaspartic residues to produce 2 subunits, large and small, that dimerizeto form the active enzyme. This protein was shown to cleave and activatecaspases 6, 7 and 9, and itself could be processed by caspases 8, 9 and10. Caspases are initially expressed as zymogens, in which a largesubunit is N-terminal to a small subunit. Caspases are generallyactivated by cleavage at internal Asp residues. Caspases are found in amyriad of organisms, including human, mouse, insect (e.g., Drosophila),and other invertebrates (e.g., C. elegans). The caspases include, butare not limited to, Caspase-1 (also known as “ICE”), Caspase-2 (alsoknown as “ICH-1”), Caspase-3 (also known as “CPP32,” “Yama,” “apopain”),Caspase-4 (also known as “ICE._(relll)”; “TX,” “ICH-2”), Caspase-5 (alsoknown as “ICE._(rellll)”; “TY”), Caspase-6 (also known as “Mch2”),Caspase-7 (also known as “Mch3,” “ICE-LAP3” “CMH-1”), Caspase-8 (alsoknown as “FLICE;” “MACH;” ‘Mch5”), Caspase-9 (also known as “ICE-LAP6;”“Mch6”), Caspase-10 (also known as “Mch4,” “FLICE-2”).

The term “apoptosis inhibiting compound” as used herein refers to anagent that can reduce apoptosis by a detectable amount by acting on apathway involved in apoptosis. The agent can act by inhibiting orblocking a particular step in the apoptotic pathway, for example byblocking or inhibiting the activity of protein involved in the pathway.The apoptotic pathway includes both caspase-dependent apoptosis, as wellas caspase-independent apoptosis.

The term “inhibit” or “inhibiting” as used herein refers to a measurablereduction of apoptotic activity that leads to at least a 10% preferablyor 20%, increase in the likelihood that a cell will survive following anevent which normally causes cell death (relative to an untreated controlcell). Preferably, the cells being compared are neural cells normallysusceptible to ischemic cell death, neurodegeneration, or axotomy.Preferably, the decrease in the likelihood that a cell will die is 80%,more preferably 2-fold, most preferably, 5-fold.

The phrase “Inhibitor of Apoptosis Protein” or “IAP” is refers to anamino acid sequence which has homology to baculovirus inhibitors ofapoptosis. For example, NAIP, truncated NAIP, HIAP1, HIAP2 and XIAP arespecifically included (see U.S. Pat. No. 5,919,912; U.S. Pat. No.6,156,535; and U.S. Pat. No. 6,709,866). Preferably, such a polypeptidehas an amino acid sequence which is at least 45%, preferably 60%, andmost preferably 85% or even 95% identical to at least one of the aminoacid sequences of the NAIP, truncated NAIP, HIAP1, HIAP2, or XIAP.

The term “mutation,” as used herein, refers to any alteration of thegene encoding an inhibitor of apoptosis protein. The mutation can alterthe functionality of the protein produced by that gene. Such mutationscan include, but are not limited to, an amino acid substitution whereina native amino acid is replaced with an alanine or other biologicallycomparable amino acid residue, including, but not limited to glycine,valine, and leucine. Amino acid substitutions can be introduced into thenucleic acid sequence by standard molecular biology methods. The term“mutation” also includes a deletion or addition of nucleotide sequencesto any portion of gene that encodes an inhibitor of apoptosis protein.In one embodiment, the mutation produces a protein or fragment that nolonger binds to a caspase. In another embodiment, the mutation producesa protein or fragment that blocks the interaction of a protein such asSmac. In a preferred embodiment, the mutation produces a protein orfragment that is neuroprotective.

The term “portion” or “fragment” as used herein refers to an amino acidsequence that has fewer amino acids than the entire sequence of theinhibitor of apoptosis protein. A fragment can comprise any desireddomain, such as a BIR-3 domain. Sizes of peptide fragments can bedesigned to be less than about 200 amino acids, less than about 100amino acids, less than about 80 amino acids, less than about 60 aminoacids, less than about 40 amino acids, less than about 20 amino acids,and less than about 10 amino acids, so long as the peptide fragmentretains a desired activity.

The term “modulate” or “modify” are used interchangeably herein andrefer to an alleviation of at least one target protein or gene involvedin the caspase-dependent pathway for apoptosis. Such that apoptosis isinhibited or reduced. A modification in apoptosis can be assessed bymonitoring cell blebbing DNA fragmentation, and the like.

The terms “neurological disorder” or “neurodegenerative disorder” areused interchangeably herein and refer to an impairment or absence of anormal neurological function or presence of an abnormal neurologicalfunction in a subject. For example, neurological disorders can be theresult of disease, injury, and/or aging. As used herein, neurologicaldisorder also includes neurodegeneration which causes morphologicaland/or functional abnormality of a neural cell or a population of neuralcells. Non-limiting examples of morphological and functionalabnormalities include physical deterioration and/or death of neuralcells, abnormal growth patterns of neural cells, abnormalities in thephysical connection between neural cells, under- or over production of asubstance or substances, e.g., a neurotransmitter, by neural cells,failure of neural cells to produce a substance or substances which itnormally produces, production of substances, e.g., neurotransmitters,and/or transmission of electrical impulses in abnormal patterns or atabnormal times. Neurodegeneration can occur in any area of the brain ofa subject and is seen with many disorders including, for example,Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, Huntington'sdisease, Parkinson's disease, and Alzheimer's disease.

The term “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount of theproteasome modulating pharmacological agent may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the pharmacological agent to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thepharmacological agent are outweighed by the therapeutically beneficialeffects.

The term “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The term “subject” as used herein refers to any living organism capableof eliciting an immune response. The term subject includes, but is notlimited to, humans, nonhuman primates such as chimpanzees and other apesand monkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

The invention is described in more detail in the following subsections:

I. Neurodegenerative Diseases

Evidence is accumulating that as a result of the normal aging processthe body increasingly loses the ability to adequately degrade mutated ormisfolded proteins. The proteasome is the piece of biological machinerythat is responsible for most normal degradation of proteins found insidecells. Age-related loss of function, or change in function of theproteasome is now thought to be at the heart of many neurodegenerativeconditions, including, for example, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, Multiple Sclerosis and amyotrophiclateral sclerosis (ALS), each of which is described below.

(a) Huntington's Disease

Huntington's disease (HD) is a hereditary disorder caused by thedegeneration of neurons in certain areas of the brain. This degenerationis genetically programmed to occur in certain areas of the brain,including the cells of the basal ganglia, the structures that areresponsible for coordinating movement. Within the basal ganglia,Huntington's disease specifically targets nerve cells in the striatum,as well as cells of the cortex, or outer surface of the brain, whichcontrol thought, perception and memory. Neuron degeneration due to HDcan result in uncontrolled movements, loss of intellectual capacity andfaculties, and emotional disturbance, such as, for example, mood swingsor uncharacteristic irritability or depression.

As discussed above, neuron degeneration due to HD is geneticallyprogrammed to occur in certain areas of the brain. Studies have shownthat Huntington's disease is caused by a genetic defect on chromosome 4,and in particular, people with HD have an abnormal repetition of thegenetic sequence CAG in the HD gene, which has been termed IT15. TheIT15 gene is located on the short arm of chromosome 4 and encodes aprotein called huntingtin. Exon I of the IT15 gene contains apolymorphic stretch of consecutive glutamine residues, known as thepolyglutamine tract (D. Rubinsztein, “Lessons from Animal Models ofHuntington's Disease,” TRENDS in Genetics, 18(4): 202-9 (April 2002)).Asymptomatic individuals typically contain fewer than 35 CAG repeats inthe polyglutamine tract.

The inherited mutation in HD is an expansion of the natural CAG repeatswithin the sequence of exon 1 of the human HD gene. This leads to anabnormally long stretch of polyglutamines. The length of thepolyglutamine repeats correlates with the severity of the disease. Oneof the pathological hallmarks of HD is a buildup of intracellularprotein aggregates composed of these abnormal HD proteins with longpolyglutamine repeats. The results in the Examples section show thatexpression of this abnormal HD gene (called Huntington) in culturedneurons leads to cell death, while co-expression of the anti-apoptoticgene XIAP blocks this death. This demonstrates that expression of ananti-apoptotic gene can protect from mutant Huntington-induced neuronaldeath.

(b) Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic disease that is characterized by“attacks,” during which areas of white matter of the central nervoussystem, known as plaques, become inflamed. Inflammation of these areasof plaque is followed by destruction of myelin, the fatty substance thatforms a sheath or covering that insulates nerve cell fibers in the brainand spinal cord. Myelin facilitates the smooth, high-speed transmissionof electrochemical messages between the brain, spinal cord, and the restof the body. Damage to the myelin sheath can slow or completely blockthe transmission of these electrochemical messages, which can result indiminished or lost bodily function.

The most common course of MS manifests itself as a series of attacks,which are followed by either complete or partial remission, during whichthe symptoms lessen only to return at some later point in time. Thistype of MS is commonly referred to as “relapsing-remitting MS.” Anotherform of MS, called “primary-progressive MS,” is characterized by agradual decline into the disease state, with no distinct remissions andonly temporary plateaus or minor relief from the symptoms. A third formof MS, known as “secondary-progressive MS,” starts as arelapsing-remitting course, but later deteriorates into aprimary-progressive course of MS.

The symptoms of MS can be mild or severe, acute or of a long duration,and may appear in various combinations. These symptoms can includevision problems such as blurred or double vision, red-green colordistortion, or even blindness in one eye, muscle weakness in theextremities, coordination and balance problems, muscle spasticity,muscle fatigue, paresthesias, fleeting abnormal sensory feelings such asnumbness, prickling, or “pins and needles” sensations, and in the worstcases, partial or complete paralysis. About half of the people sufferingfrom MS also experience cognitive impairments, such as for example, poorconcentration, attention, memory and/or judgment. These cognitivesymptoms occur when lesions develop in those areas of the brain that areresponsible for information processing.

(c) Alzheimer's Disease

Alzheimer's disease is a progressive, neurodegenerative disease thataffects the portions of the brain that control thought, memory andlanguage. This disease is characterized by progressive dementia thateventually results in substantial impairment of both cognition andbehavior. The disease manifests itself by the presence of abnormalextracellular protein deposits in brain tissue, known as “amyloidplaques,” and tangled bundles of fibers accumulated within the neurons,known as “neurofibrillary tangles,” and by the loss of neuronal cells.The areas of the brain affected by Alzheimer's disease can vary, but theareas most commonly affected include the association cortical and limbicregions.

Symptoms of Alzheimer's disease include memory loss, deterioration oflanguage skills, impaired visuospatial skills, and impaired judgment,yet those suffering from Alzheimer's retain motor function.

(d) Parkinson's Disease

Parkinson's disease (PD) is characterized by death of dopaminergicneurons in the substantia nigra (SNr), leading to a disturbance in thebasal ganglia network which regulates movement. In addition, otherbrainstem cell populations can die or become dysfunctional. One of thepathological hallmarks of PD in humans is the Lewy body, which containsabnormal protein aggregates which include the protein alpha-synuclein.While there are many therapies available to treat the symptoms ofParkinson's disease, including medical therapy and surgical therapies,there is no current treatment which will stop the death of neurons andultimately cure this disorder.

To date, the cause of neuronal death has remained elusive. One problemhas been the relevance of current animal models to human disease. Thegold-standard animal models for PD involve rapid destruction of dopamineneurons using chemicals which are fairly specific for dopamine neurons.These chemical toxins, which include 6-hydroxydopamine (6OHDA) and MPTP,cause oxidative damage to dopamine neurons in both rodents and primates.These models can be useful to test the efficacy of new therapiesdesigned to improve the symptoms of PD, since such treatments aredesigned to intervene after cells have died or become dysfunctional,regardless of the cause of cell death. In order to test the value ofprotective or curative strategies, however, the mechanism of cell deathmust be relevant to human disease otherwise successful experimentalstudies will not translate into effective human therapy.

Many features of the animal models have been questioned for protectivestrategies. First, these toxins usually cause near complete destructionof dopamine neurons within 24-48 hrs., while PD is a slowly degenerativedisease which can take many years or more to have even partial loss ofcells. Also, these do not cause protein inclusions similar to the Lewybodies seen in human PD. These toxins are also only specific to dopamineneurons, while in human PD other cell populations are affected. There isalso little convincing evidence in human disease that the oxidativedamage mechanism is the primary cause of PD. Nonetheless, severalfactors have been shown to protect animal cells from these toxins,including anti-apoptotic genes and growth factors such as glial-derivedneurotrophic factor (GDNF). This is understandable, since the result ofsuch oxidative damage is usually apoptotic cell death.

The history of GDNF highlights the problems in translating promisingdata from these models to human disease. Several animal studies overmany years suggested that GDNF could afford substantial protection todopamine neurons when exposed to either 6OHDA or MPTP. Similar data hasbeen obtained regardless of the mode of delivery of GDNF, including bothintraventricular and direct intrastriatal infusion of recombinant GDNFprotein, as well as GDNF produced from a viral vector following genetherapy. Nonetheless, multiple GDNF studies in human have failed. Thefirst studies involved infusion of GDNF into cerebrospinal fluid via anintraventricular catheter. This was stopped due to adverse effects. Itwas then hypothesized that direct infusion of GDNF into the striatum,where dopamine neuron terminals reside, would limit side effects andimprove efficacy as was seen in the above mentioned animal models. Thiswas also recently halted due to failure to demonstrate any meaningfuleffect in human patients compared to controls. This only serves tohighlight problems with developing neuroprotective therapeutics usingthese models. In fact, the only similarity between these models andhuman PD is the loss of dopamine neurons. This, however, can also beachieved by many other means, including thermal destruction ordestruction of these cells using other chemicals such as ibotinic acid.Therefore, there is no good evidence that any protection of neuronsusing these models has any value to human PD.

Recently, a new model was described which not only appears to be morerelevant to human PD, but which also is consistent with most of theknown features of human disease (Kevin et al, Annals of Neurology (2004)56, 149-162). The model involves repeated administration of a proteasomeinhibitor. Proteasomes are complex, multi-unit enzymes within the cellwhich are critical for metabolizing and removing proteins which aremisfolded, dysfunctional and/or no longer desirable. These are essentialfor protein turnover, which is crucial for proper regulation of cellularphysiology. Proteins which are targeted to the proteasome are usuallymodified by addition of a ubiquitin group. Ubiquinated proteins can thenenter the proteasome for ultimate degradation. Unlike the dopamine toxinmodel, this model causes a very slow neuronal degeneration which is muchmore analogous to human disease. In addition to dopamine neuronal lossin the SNr, loss or dysfunctional of other neuronal populations are seenwhich also mimic the human disorder. Most interestingly, intracellularprotein aggregates are seen which are highly analogous to the Lewy body.None of these features are present in the dopamine toxin models, and allof them are found to some degree in the human disorder, indicating thatthis is a far more relevant model of the actual mechanism of cell deathin human PD.

Those few forms of human PD for which a cause is known further supportthe relevance of this model for neuroprotection studies. A minority ofPD cases are caused by inherited mutations in a single gene. To date,four such genes have been identified. While the function of one generemains unknown, the other three directly support the concept thatubiquitin-proteasome dysfunction is the key cause of cell death PD. Twoof these genes, parkin and UCHL-1, are involved in ubiquination ofproteins and loss of function causes human PD. The third gene,alpha-synuclein, causes a dominant form of PD and, as mentioned earlier,is a key component of the intracellular inclusions called Lewy bodies.Therefore, the major known causes of inherited human PD support thepathological findings in the new proteasome inhibitor model of PD asbeing the only available model which accurately replicates the humandisorder.

The factors triggering dopaminergic neuronal loss in Parkinson's diseaseare still largely unknown. Neuronal cell death is thought to occur viaapoptosis and the involvement of several caspases at the late stages ofthis process is well documented. X-linked inhibitor of apoptosis (XIAP)is a potent inhibitor of caspases 9, 3 and 7 and thus an attractivecandidate as a potentially therapeutic neuroprotective factor.

The effects of XIAP in several in vitro models of Parkinson's diseasewas examined and the results shown in the Example section. Most of theexperiments were performed using a more stable truncated from of XIAPlacking the RING domain at the C-terminus (dXIAP). To test the in vivoeffect of XIAP, a recombinant AAV (rAAV) vector expressing dXIAP wasgenerated to investigate therapeutic intervention in the novel in vivorat model of Parkinson's disease which is triggered by inhibition ofproteasome machinery. The data demonstrates that dXIAP may be used forneuroprotection.

(e) Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a universally fatalneurodegenerative condition in which patients progressively lose allmotor function—unable to walk, speak, or breathe on their own, ALSpatients die within two to five years of diagnosis. The incidence of ALSincreases substantially in the fifth decade of life. Evidence isaccumulating that as a result of the normal aging process the bodyincreasingly loses the ability to adequately degrade mutated ormisfolded proteins. The proteasome is the piece of biological machineryresponsible for most normal degradation of proteins inside cells. Agerelated loss of function or change of function of the proteasome is nowthought to be at the heart of many neurodegenerative conditions,including Alzheimer's disease, Parkinson's disease, Huntington'sdisease, and ALS.

The cardinal feature of ALS is the loss of spinal motor neurons, whichcauses the muscles under their control to weaken and waste away leadingto paralysis. ALS has both familial (5-10%) and sporadic forms and thefamilial forms have now been linked to several distinct genetic loci(Deng, H. X., et al., “Two novel SOD1 mutations in patients withfamilial amyotrophic lateral sclerosis,” Hum. Mol. Genet., 4(6): 1113-16(1995); Siddique, T. and A. Hentati, “Familial amyotrophic lateralsclerosis,” Clin. Neurosci., 3(6): 338-47 (1995); Siddique, T., et al.,“Familial amyotrophic lateral sclerosis,” J. Neural Transm. Suppl., 49:219-33 (1997); Ben Hamida, et al., “Hereditary motor system diseases(chronic juvenile amyotrophic lateral sclerosis). Conditions combining abilateral pyramidal syndrome with limb and bulbar amyotrophy,” Brain,113(2): 347-63 (1990); Yang, Y., et al., “The gene encoding alsin, aprotein with three guanine-nucleotide exchange factor domains, ismutated in a form of recessive amyotrophic lateral sclerosis,” Nat.Genet., 29(2): 160-65 (2001); Hadano, S., et al., “A gene encoding aputative GTPase regulator is mutated in familial amyotrophic lateralsclerosis 2,” Nat. Genet., 29(2): 166-73 (2001)). About 15-20% offamilial cases are due to mutations in the gene encoding Cu/Znsuperoxide dismutase 1 (SOD1) (Siddique, T., et al., “Linkage of a genecausing familial amyotrophic lateral sclerosis to chromosome 21 andevidence of genetic-locus heterogeneity,” N. Engl. J. Med., 324(20):1381-84 (1991); Rosen, D. R., et al., “Mutations in Cu/Zn superoxidedismutase gene are associated with familial amyotrophic lateralsclerosis.” Nature, 362(6415): 59-62 (1993)).

Although the etiology of the disease is unknown, the dominant theory isthat neuronal cell death in ALS is the result of over-excitement ofneuronal cells due to excess extracellular glutamate. Glutamate is aneurotransmitter that is released by glutaminergic neurons, and is takenup into glial cells where it is converted into glutamine by the enzymeglutamine synthetase, glutamine then re-enters the neurons and ishydrolyzed by glutaminase to form glutamate, thus replenishing theneurotransmitter pool. In a normal spinal cord and brain stem, the levelof extracellular glutamate is kept at low micromolar levels in theextracellular fluid because glial cells, which function in part tosupport neurons, use the excitatory amino acid transporter type 2(EAAT2) protein to absorb glutamate immediately. A deficiency in thenormal EAAT2 protein in patients with ALS, was identified as beingimportant in the pathology of the disease (See e.g., Meyer et al., J.Neurol. Neurosurg. Psychiatry, 65: 594-596 (1998); Aoki et al., Ann.Neurol. 43: 645-653 (1998); Bristol et al., Ann Neurol. 39: 676-679(1996)). One explanation for the reduced levels of EAAT2 is that EAAT2is spliced aberrantly (Lin et al., Neuron, 20: 589-602 (1998)). Theaberrant splicing produces a splice variant with a deletion of 45 to 107amino acids located in the C-terminal region of the EAAT2 protein (Meyeret al., Neureosci Lett. 241: 68-70 (1998)). Due to the lack of, ordefectiveness of EAAT2, extracellular glutamate accumulates, causingneurons to fire continuously. The accumulation of glutamate has a toxiceffect on neuronal cells because continual firing of the neurons leadsto early cell death.

II. Proteasomes and Proteasome Modulation

In one aspect, the invention pertains to using an inhibitor of apoptosisprotein (TAP), e.g., XIAP, for the amelioration or treatment ofneurological and/or neurodegenerative disorders and diseases associatedwith abnormal proteasome function.

The proteasome is a multi-unit protein complex that plays a key role inprotein degradation within a cell. The function of this key processranges from ridding the cell of old and misfolded proteins to thedegradation of key regulatory proteins and antigen generation for immunesurveillance. In particular, proteolysis is involved in the regulationof numerous cellular processes including progression of the cell cycle,oncogenesis, transcription, development, growth and atrophy of developedtissues, flow of substrates through metabolic pathways, selectiveelimination of abnormal proteins and antigen processing (DeMartino, G.,et al., “The Proteasome, a Novel Protease Regulated by MultipleMechanisms,” J. Biol. Chem., 274(32): 22123-126 (1999); Ottosen, S., etal., “Protease Parts at Gene Promoters,” Science, 296: 479-81 (2002)).The antigen-generating function of the proteasome allows targetedkilling of defective and virally infected cells by the Cytotoxic T-cellsand Natural killer cells.

The proteasome undergoes extensive modification to suit its differentfunction. It does so by adding and replacing the individual subunits andby restructuring. At the core of all configurations is the 20Sproteasome, which provides the proteasome its catalytic degradationpower. 20S proteasomes are combined with various regulatory caps such asPA700 and PA28, which are thought to control the entry to 20S as well asthe disposition of end products. The core of the 20S proteasome consistsof two copies each of seven different α and β subunits, which arearranged in four stacked rings (α₇β₇β₇α₇) (Verma et al., “ProteasomalProteomics: Identification of Nucleotide-sensitiveProteasome-interacting Proteins by Mass Spectrometric Analysis ofAffinity-purified Proteasomes,”Mol. Biol. Cell., 11: 3425-39 (2000)).The interior of the ring structure contains a cavity consisting of threecontiguous chambers joined by narrow constrictions (DeMartino, G., etal., “The Proteasome, a Novel Protease Regulated by MultipleMechanisms,” J. Biol. Chem., 274(32): 22123-126 (1999)). The 7 betasubunits of the 20S proteasome provide the bulk of its peptide cleavingabilities. Three of these subunits, X (β5), Y (β1), and Z (β2) can bereplaced with inducible counterparts LMP2, LMP7, and MECL-1, whichcauses the proteasome to cleave peptides in a manner more specific forMHC I antigen presentation (Toes, R. E., et al., “Discrete cleavagemotifs of constitutive and immunoproteasomes revealed by quantitativeanalysis of cleavage products,” J. Exp. Med., 194(1): 1-12 (2001)).These proteins are selectively induced under certain conditions,including treatment of cells with gamma-interferon. The LMP2, LMP7 andMECL-1 subunits assembly to form proteasomes with distinct subunitcompositions and altered catalytic characteristics (DeMartino, G., etal., “The Proteasome, a Novel Protease Regulated by MultipleMechanisms,” J. Biol. Chem., 274(32): 22123-126 (1999)). Thisconfiguration is known as the ‘immunoproteasome’ and is commonlypresented in response to viral infection.

Increasing evidence is accumulating that as a result of the normal agingprocess the body increasingly loses the ability to adequately degrademutated or misfolded proteins. The proteasome is the cell machineryresponsible for normal protein degradation. Oxidative stress is thoughtto contribute to this process of protein degradation through oxidationand nitration of intracellular proteins, which makes proteins prone tocross-linking and aggregation (Davies, K. J., “Degradation of oxidizedproteins by the 20S proteasome,” Biochimie, 83(3-4): 301-10 (2001);Squier, T. C., “Oxidative stress and protein aggregation duringbiological aging,” Exp. Gerontol., 36(9): 1539-50 (2001)). Suchaggregated proteins are more resistant to degradation in the proteasomeand may cause inhibition of proteasomal function through irreversiblebinding to the proteasome (Davies, K. J., “Degradation of oxidizedproteins by the 20S proteasome,” Biochimie, 83(3-4): 301-10 (2001);Squier, T. C., “Oxidative stress and protein aggregation duringbiological aging,” Exp. Gerontol., 36(9): 1539-50 (2001)). Alternativelyor additionally, decreased proteasomal activity may be caused moredirectly by oxidation of the proteasome itself (Keller, J. N, et al.,“Possible involvement of proteasome inhibition in aging: implicationsfor oxidative stress,”Mech. Ageing Dev., 113(1): 61-70 (2000)).Aggregates of misfolded proteins can induce a number of changes in theproteasome that can lead to aberrant immune activation and apoptoticcell death. Age related loss of function or impediment of the proteasomeis now thought to be at the heart of many neurodegenerative conditionssuch as Alzheimer's disease, Parkinson's disease, Huntington's disease,and ALS (Goldberg, et al., “The cellular chamber of doom,” Sci. Am.,284(1): 68-73 (2001); Johnston, J. A., et al., “Formation of highmolecular weight complexes of mutant Cu, Zn-superoxide dismutase in amouse model for familial amyotrophic lateral sclerosis,” Proc. Natl.Acad. Sci. USA, 97(23): 12571-76 (2000); Kopito, R. R., “Aggresomes,inclusion bodies and protein aggregation,” Trends Cell. Biol., 10(12):524-30 (2000)).

Inhibition of proteasomal activity increases abnormal proteinaccumulation, and accumulation of abnormal proteins contribute toinhibition of proteasomal activity. Proteasomal inhibition is a commonfeature in neurodegenerative diseases. Accordingly, proteasomaldysfunction can alter the progression of diseases such as Parkinson'sdisease and Huntington's disease by a variety of ways. It is believedthat proteasome alteration modulates important factors involved in cellcycle regulation, apoptosis, inflammation, and antigen presentation,which individually or in combination can lead to disease propagation.

III. Apoptosis and Inhibitor of Apoptosis Proteins

In one aspect, the invention pertains to reducing, inhibiting,preventing or altering apoptosis associated with abnormal proteasomefunction using an inhibitor of apoptosis protein (TAP), e.g., XAIP.Apoptosis or programmed cell death was originally described by Kerr etal., in 1972 (Kerr, et al. (1972) Br J Cancer, 26, 239-57), as a newcell-autonomous mechanism of death aimed at removing damaged, mutated oraged cells. Mitochondria, are directly linked to the process ofapoptosis to the extent that they are now termed “killer organelles”(Ravagnan, et al. (2002) J Cell Physiol, 192, 131-7). Mitochondriacontain several proteins in their inter-membrane space which arereleased in response to pro-apoptotic stimuli. These diverse proteinsinclude cytochrome c, AIF, Smac/Diablo, endonuclease G and Omi/HtrA2(See e.g., Van Loo, et al. (2002) Cell Death Differ, 9, 1031-42). Eachof these proteins has a distinct function but they are all pro-apoptoticand, invariably, their release from the mitochondria induces cell death.

Several gene products that modulate the apoptotic process have beenidentified. Although these products can, in general, be separated intotwo basic categories, gene products from each category can function toeither inhibit or induce apoptosis. One family of gene products is theBcl-2 family of proteins. A second family of gene products, the caspasefamily, is related genetically to the C. elegans ced-3 gene product,which is required for apoptosis in the roundworm, C. elegans. Thecaspase family includes, for example, caspase-1, caspase-2, caspase-3,caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9 andcaspase-10.

Examples of inhibitor of apoptosis protein include, but are not limitedto, X-linked inhibitor of apoptosis protein (XIAP), NIAP, cIAP-1 andcIAP-2.

In the gene therapy constructs, an IAP cDNA expression is directed fromany suitable promoter (e.g., the human cytomegalovirus, simian virus 40,or metallothionein promoters), and its production is regulated by anydesired mammalian regulatory element. For example, if desired, enhancersknown to direct preferential gene expression in neuronal cells may beused to direct expression of an IAP.

Fragments or derivatives of the IAP, e.g., XIAP, may also beadministered by retroviral gene transfer therapy or another suitableviral vector system. Useful fragments or derivatives of IAP, e.g., XIAPmay be administered by inserting the nucleic acids encoding thesefragments or derivatives in place of the complete IAP, e.g., XIAP genein a gene therapy vector. The sequence of IAP proteins and nucleic acidsmay be obtained from U.S. Pat. No. 5,919,912; U.S. Pat. No. 6,156,535;and U.S. Pat. No. 6,709,866. The fragment can comprise any desireddomain, such as a BIR-3 domain. Sizes of peptide fragments can be lessthan about 200 amino acids, less than about 100 amino acids, less thanabout 80 amino acids, less than about 60 amino acids, less than about 40amino acids, less than about 20 amino acids, and less than about 10amino acids, so long as the peptide fragment retains a desired activity.

The peptides fragments can be chemically synthesized using a peptidesynthesizer based on the available sequences. These chemicallysynthesized fragments can be synthesized to include a mutation. Thepeptide fragments can also be mutated using standard molecular biologytechniques. With this approach, the nucleotide sequence encoding thepeptide fragment is altered by insertion, deletion or addition ofnucleotides.

If the peptides are chemically synthesized, they can be modified byadding signal peptides or other regulatory sequences. Signal peptidesplay an important role in protein transport and sorting to the differentcompartment of the cell. Although signal peptides have varying lengthsand do not have a consensus sequence, almost all possess a commonthree-region structure: the positively charged n-region, the hydrophobicN-region, and the C-region where the cleavage site occurs (Nakai, (2000)Adv. in Protein Chem. 54:277-344). Signal peptides are cleaved whileproteins are still being processed. Examples of signal peptides include,but are not limited to, N-terminus signal peptides that often target themitochondrial matrix; and C-terminus signal peptides that functionsimilarly to N-terminus signal peptides, and Suomen Kristillinen Liitto(SKL). If the IAP peptides are being expressed, the nucleotide sequencethat encodes a signal peptide can be coupled to the nucleotide sequenceencoding the IAP peptide.

The nucleotide sequence encoding the IAP peptides can also be modifiedby adding transactivating regulatory protein (TAT) protein of HIV actsas transcriptional regulator of viral gene expression by binding to thetransactivating responsive sequence (TAR) RNA element. Binding to theTAR RNA element initiates viral transcription and/or elongation from LTRpromoter (Feng (1988) Nature 334: 165-167 and Roy (1990) Genes Dev 4:1365); upregulates expression of all viral genes; promotes theelongation phase of HIV-1 transcription, allowing full-lengthtranscripts to be produced; and represses cellular promoters.

IV. Vectors

The vectors of the invention can be delivered to the cells of thecentral nervous system by using viral vectors or by using non-viralvectors. In a preferred embodiment, the invention uses adeno-associatedviral (AAV) vectors comprising the a nucleotide sequence encoding IAPfor gene delivery. AAV vectors can be constructed using known techniquesto provide at least the operatively linked components of controlelements including a transcriptional initiation region, a exogenousnucleic acid molecule, a transcriptional termination region and at leastone post-transcriptional regulatory sequence. The control elements areselected to be functional in the targeted cell. The resulting constructwhich contains the operatively linked components is flanked at the 5′and 3′ region with functional AAV ITR sequences.

The nucleotide sequences of AAV ITR regions are known. The ITR sequencesfor AAV-2 are described, for example by Kotin et al. (1994) Human GeneTherapy 5:793-801; Berns “Parvoviridae and their Replication” inFundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.)The skilled artisan will appreciate that AAV ITR's can be modified usingstandard molecular biology techniques. Accordingly, AAV ITRs used in thevectors of the invention need not have a wild-type nucleotide sequence,and may be altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, AAV ITRs may be derived from any of severalAAV serotypes, including but not limited to, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, and the like. Furthermore, 5′ and 3′ ITRs which flank aselected nucleotide sequence in an AAV expression vector need notnecessarily be identical or derived from the same AAV serotype orisolate, so long as the ITR's function as intended, i.e., to allow forexcision and replication of the bounded nucleotide sequence of interestwhen AAV rep gene products are present in the cell.

The skilled artisan can appreciate that regulatory sequences can oftenbe provided from commonly used promoters derived from viruses such as,polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Use of viralregulatory elements to direct expression of the protein can allow forhigh level constitutive expression of the protein in a variety of hostcells. Ubiquitously expressing promoters can also be used include, forexample, the early cytomegalovirus promoter Boshart et al. (1985) Cell41:521-530, herpesvirus thymidine kinase (HSV-TK) promoter (McKnight etal. (1984) Cell 37: 253-262), β-actin promoters (e.g., the human β-actinpromoter as described by Ng et al. (1985) Mol. Cell. Biol. 5: 2720-2732)and colony stimulating factor-1 (CSF-1) promoter (Ladner et al. (1987)EMBO J. 6: 2693-2698).

Alternatively, the regulatory sequences of the AAV vector can directexpression of the gene preferentially in a particular cell type, i.e.,tissue-specific regulatory elements can be used. Non-limiting examplesof tissue-specific promoters which can be used include, central nervoussystem (CNS) specific promoters such as, neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477) and glial specific promoters (Morii et al.(1991) Biochem. Biophys Res. Commun. 175: 185-191). Preferably, thepromoter is tissue specific and is essentially not active outside thecentral nervous system, or the activity of the promoter is higher in thecentral nervous system that in other systems. For example, a promoterspecific for the spinal cord, brainstem, (medulla, pons, and midbrain),cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpusstratium, cerebral cortex, or within the cortex, the occipital,temporal, parietal or frontal lobes), or combinations, thereof. Thepromoter may be specific for particular cell types, such as neurons orglial cells in the CNS. If it is active in glial cells, it may bespecific for astrocytes, oligodentrocytes, ependymal cells, Schwanncells, or microglia. If it is active in neurons, it may be specific forparticular types of neurons, e.g., motor neurons, sensory neurons, orinterneurons. Preferably, the promoter is specific for cells inparticular regions of the brain, for example, the cortex, stratium,nigra and hippocampus.

Suitable neuronal specific promoters include, but are not limited to,CMV/CBA, neuron specific enolase (NSE) (Olivia et al. (1991) Genomics10: 157-165, GenBank Accession No: X51956), and human neurofilamentlight chain promoter (NEFL) (Rogaev et al. (1992) Hum. Mol. Genet. 1:781, GenBank Accession No: L04147). Glial specific promoters include,but are not limited to, glial fibrillary acidic protein (GFAP) promoter(Morii et al. (1991) Biochem. Biophys Res. Commun. 175: 185-191, GenBankAccession No:M65210), 5100 promoter (Morii et al. (1991) Biochem.Biophys Res. Commun. 175: 185-191, GenBank Accession No: M65210) andglutamine synthase promoter (Van den et al. (1991) Biochem. Biophys.Acta. 2: 249-251, GenBank Accession No: X59834). In a preferredembodiment, the gene is flanked upstream (i.e., 5′) by the neuronspecific enolase (NSE) promoter. In another preferred embodiment, thegene of interest is flanked upstream (i.e., 5′) by the elongation factor1 alpha (EF) promoter.

The AAV vector harboring the nucleotide sequence encoding a protein ofinterest, e.g., GAD, and a post-transcriptional regulatory sequence(PRE) flanked by AAV ITRs, can be constructed by directly inserting thenucleotide sequence encoding the protein of interest and the PRE into anAAV genome which has had the major AAV open reading frames (“ORFs”)excised therefrom. Other portions of the AAV genome can also be deleted,as long as a sufficient portion of the ITRs remain to allow forreplication and packaging functions.

These constructs can be designed using techniques well known in the art.(See, e.g., Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka(1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin(1994) Human Gene Therapy 5:793-801; Shelling et al. (1994) Gene Therapy1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875).

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook et al., Supra. SeveralAAV vectors are available from the American Type Culture Collection(“ATCC”) under Accession Numbers 53222, 53223, 53224, 53225 and 53226.

In order to produce recombinant AAV particles, an AAV vector can beintroduced into a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook etal. (1989) Molecular Cloning, a laboratory manual, Cold Spring HarborLaboratories, N.Y., Davis et al. (1986) Basic Methods in MolecularBiology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularlysuitable transfection methods include calcium phosphate co-precipitation(Graham et al. (1973) Virol. 52:456-467), direct micro-injection intocultured cells (Capecchi (1980) Cell 22:479-488), electroporation(Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediatedgene transfer (Mannino et al. (1988) BioTechniques 6:682-690),lipid-mediated transduction (Felgner et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocitymicroprojectiles (Klein et al. (1987) Nature 327:70-73).

Suitable host cells for producing recombinant AAV particles include, butare not limited to, microorganisms, yeast cells, insect cells, andmammalian cells, that can be, or have been, used as recipients of aexogenous nucleic acid molecule. Thus, a “host cell” as used hereingenerally refers to a cell which has been transfected with an exogenousnucleic acid molecule. The host cell includes any eukaryotic cell orcell line so long as the cell or cell line is not incompatible with theprotein to be expressed, the selection system chosen or the fermentationsystem employed. Non-limiting examples include CHO dhfr-cells (Urlauband Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220), 293 cells(Graham et al. (1977) J. Gen. Virol. 36: 59) or myeloma cells like SP2or NS0 (Galfre and Milstein (1981) Meth. Enzymol. 73(B):3-46).

In one embodiment, cells from the stable human cell line, 293 (readilyavailable through, e.g., the ATCC under Accession No. ATCC CRL1573) arepreferred in the practice of the present invention. Particularly, thehuman cell line 293, which is a human embryonic kidney cell line thathas been transformed with adenovirus type-5 DNA fragments (Graham et al.(1977) J. Gen. Virol. 36:59), and expresses the adenoviral E1a and E1bgenes (Aiello et al. (1979) Virology 94:460). The 293 cell line isreadily transfected, and provides a particularly convenient platform inwhich to produce rAAV virions.

Host cells containing the above-described AAV vectors must be renderedcapable of providing AAV helper functions in order to replicate andencapsidate the expression cassette flanked by the AAV ITRs to producerecombinant AAV particles. AAV helper functions are generallyAAV-derived coding sequences which can be expressed to provide AAV geneproducts that, in turn, function in trans for productive AAVreplication. AAV helper functions are used herein to complementnecessary AAV functions that are missing from the AAV vectors. Thus, AAVhelper functions include one, or both of the major AAV open readingframes (ORFs), namely the rep and cap coding regions, or functionalhomologues thereof.

The AAV rep coding region of the AAV genome encodes the replicationproteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expressionproducts have been shown to possess many functions, includingrecognition, binding and nicking of the AAV origin of DNA replication,DNA helicase activity and modulation of transcription from AAV (or otherexogenous) promoters. The Rep expression products are collectivelyrequired for replicating the AAV genome. The AAV cap coding region ofthe AAV genome encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. AAV helper functions can be introducedinto the host cell by transfecting the host cell with an AAV helperconstruct either prior to, or concurrently with, the transfection of theAAV vector comprising the expression cassette, AAV helper constructs arethus used to provide at least transient expression of AAV rep and/or capgenes to complement missing AAV functions that are necessary forproductive AAV infection. AAV helper constructs lack AAV ITRs and canneither replicate nor package themselves. These constructs can be in theform of a plasmid, phage, transposon, cosmid, virus, or virion. A numberof AAV helper constructs have been described, such as the commonly usedplasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expressionproducts. (See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; andMcCarty et al. (1991) J. Virol. 65:2936-2945). A number of other vectorshave been described which encode Rep and/or Cap expression products.See, e.g., U.S. Pat. No. 5,139,941.

As a consequence of the infection of the host cell with a helper virus,the AAV Rep and/or Cap proteins are produced. The Rep proteins alsoserve to duplicate the AAV genome. The expressed Cap proteins assembleinto capsids, and the AAV genome is packaged into the capsids. Thisresults the AAV being packaged into recombinant AAV particles comprisingthe expression cassette. Following recombinant AAV replication,recombinant AAV particles can be purified from the host cell using avariety of conventional purification methods, such as CsCl gradients.The resulting recombinant AAV particles are then ready for use for genedelivery to various cell types.

Alternatively, a vector of the invention can be a virus other than theadeno-associated virus, or portion thereof, which allows for expressionof a nucleic acid molecule introduced into the viral nucleic acid. Forexample, replication defective retroviruses, adenoviruses and lentiviruscan be used. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel et al. (eds.) GreenePublishing Associates, (1989), Sections 9.10-9.14 and other standardlaboratory manuals. Examples of suitable retroviruses include pLJ, pZIP,pWE and pEM which are well known to those skilled in the art. Examplesof suitable packaging virus lines include Crip, Cre, 2 and Am. Thegenome of adenovirus can be manipulated such that it encodes andexpresses the protein of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See e.g.,Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991)Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are wellknown to those skilled in the art.

Alternatively, the vector can be delivered using a non-viral deliverysystem. This includes delivery of the vector to the desired tissues incolloidal dispersion systems that include, for example, macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes.

Liposomes are artificial membrane vesicles which are useful as deliveryvehicles in vitro and in vivo. In order for a liposome to be anefficient gene transfer vehicle, the following characteristics should bepresent: (1) encapsulation of the genetic material at high efficiencywhile not compromising the biological activity; (2) preferential andsubstantial binding to a target cell in comparison to non-target cells;(3) delivery of the aqueous contents of the vesicle to the target cellcytoplasm at high efficiency; and (4) accurate and effective expressionof genetic information (Mannino, et al. (1988) Biotechniques, 6:682).Examples of suitable lipids liposomes production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Additional examples of lipids include,but are not limited to, polylysine, protamine, sulfate and3b-[N—(N′,N′dimethylaminoethane) carbamoyl] cholesterol.

Alternatively, the vector can be coupled with a carrier for deliveryExemplary and preferred carriers are keyhole limpet hemocyanin (KLH) andhuman serum albumin. Other carriers may include a variety of lymphokinesand adjuvants such as INF, IL-2, IL-4, IL-8 and others. Means forconjugating a peptide to a carrier protein are well known in the art andinclude glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester,carbodiimyde and bis-biazotized benzidine. The vector can be conjugatedto a carrier by genetic engineering techniques that are well known inthe art. (See e.g., U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;4,599,230; 4,596,792; and 4,578,770).

In one embodiment, particle-mediated delivery using a gene-gun can beused as a method to deliver the vector. Suitable particles for genegun-based delivery of include gold particles. In one embodiment, thevector can be delivered as naked DNA. Gene gun based delivery isdescribed, for example by, Braun et al. (1999) Virology 265:46-56; Drewet al. (1999) Vaccine 18:692-702; Degano et al. (1999) Vaccine18:623-632; and Robinson (1999) Int J Mol Med 4:549-555; Lai et al.(1998) Crit. Rev Immunol 18:449-84; See e.g., Accede et al. (1991)Nature 332: 815-818; and Wolff et al. (1990) Science 247:1465-1468Murashatsu et al., (1998) Int. J. Mol. Med. 1: 55-62; Agracetus et al.(1996) J. Biotechnol. 26: 37-42; Johnson et al. (1993) Genet. Eng. 15:225-236). Also within the scope of the invention is the delivery of thevector in one or more combinations of the above delivery methods.

V. Delivery Systems

Delivery systems include methods of in vitro, in vivo and ex vivodelivery of the vector. For in vivo delivery, the vector can beadministered to a subject in a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable carrier”, as used herein, refers toany physiologically acceptable carrier for in vivo administration of thevectors of the present invention. Such carriers do not induce an immuneresponse harmful to the individual receiving the composition, and arediscussed below.

In one embodiment, vector can be distributed throughout a wide region ofthe CNS, by injecting the vector into the cerebrospinal fluid, e.g., bylumbar puncture (See e.g., Kapadia et al. (1996) Neurosurg 10: 585-587).

Alternatively, precise delivery of the vector into specific sites of thebrain, can be conducted using stereotactic microinjection techniques.For example, the subject being treated can be placed within astereotactic frame base (MRI-compatible) and then imaged using highresolution MRI to determine the three-dimensional positioning of theparticular region to be treated. The MRI images can then be transferredto a computer having the appropriate stereotactic software, and a numberof images are used to determine a target site and trajectory forantibody microinjection. The software translates the trajectory intothree-dimensional coordinates that are precisely registered for thestereotactic frame. In the case of intracranial delivery, the skull willbe exposed, burr holes will be drilled above the entry site, and thestereotactic apparatus used to position the needle and ensureimplantation at a predetermined depth. The vector can be delivered toregions, such as the cells of the spinal cord, brainstem, (medulla,pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus),telencephalon (corpus stratium, cerebral cortex, or within the cortex,the occipital, temporal, parietal or frontal lobes), or combinations,thereof. In another preferred embodiment, the vector is delivered usingother delivery methods suitable for localized delivery, such aslocalized permeation of the blood-brain barrier. Particularly preferreddelivery methods are those that deliver the vector to regions of thebrain that require modification.

VI. Pharmaceutical Compositions and Pharmaceutical Administration

The vector of the invention can be incorporated into pharmaceuticalcompositions suitable for administration to a subject. Typically, thepharmaceutical composition comprises the vector of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans. The preferred mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In one embodiment, the vector isadministered by intravenous infusion or injection. In anotherembodiment, the vector is administered by intramuscular or subcutaneousinjection. In another embodiment, the vector is administered perorally.In the most preferred embodiment, the vector is delivered to a specificlocation using stereostatic delivery.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antigen, antibody or antibody portion) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile, lyophilized powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andspray-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The vector of the present invention can be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Thepharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of the vectors of the invention. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the vector may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the vector to elicit a desired responsein the individual. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the vector are outweighed bythe therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

EXAMPLES Example 1 Methods and Materials (i) MAP Expression Plasmids

A full length human XIAP cDNA was amplified from U87-MG cells and clonedinto pcDNA4. To produce pdXIAP, C-terminal 48 amino acid comprising theRING domain were removed in a second round of PCR. The integrity of allconstructs was verified by sequencing.

(ii) Recombinant AAV Vectors

To generate AAV.dXIAP, the XIAP ORF lacking C-terminal 48 amino acid wasPCR-amplified and cloned into an AAV expression plasmid (FIG. 1). It wasengineered to contain the Kozak consensus translation start site. Acontrol vector was generated by subcloning the EGFP cDNA into the sameAAV backbone. Virus stocks were prepared by packaging the vectorplasmids into AAV cerotype 2 particles using helper-free plasmidtransfection system. The vectors were purified using heparin affinitychromatography and dialyzed against PBS. rAAV titers were determined byquantitative PCR using CMV-enhancer-specific primers and adjusted to1012 genomic particles per ml.

A schematic representation of the rAAV vectors is shown in FIG. 1. Majorelements include: AAV-2 inverted terminal repeats (ITR), hybrid CMVenhancer/chicken β-actin promoter (CMV/CBA), composite chicken β-actinpromoter/rabbit β-globin intron (CBA-RBG), enhanced green fluorescentprotein (GFP) or dXIAP, wood chuck posttranscriptional regulatoryelement (WPRE), and bovine growth hormone polyadenylation signal (BGHpoly(A)).

(iii) In Vitro Models of Parkinson's Disease

(a) 6-OHDA model. Human neuroblastoma SH-SY5Y cells were transfectedwith pcDNA4, pXIAP or pdXIAP together with pCaspase3-Sensor vector(Clonetech). In 48 h cells were treated with 70 nM 6-OHDA for 6 h,fixed, stained with propidium iodide and apoptotic cells with condensednuclei were counted. In a separate set of experiments, SH-SY5Y cellswere co-transfected with XIAP and EGFP expression plasmids for 48 h. Thecells were then treated with 50 nM 6-OHDA and apoptotic cells withnuclear YGFP fluorescence were scored 6 h later.

(b) MG-132 model. Human glioblastoma U87-MG cells were infected withAAV.GFP or AAV.dXIAP at multiplicity of infection (moi) 1,000 for 48 h.This experimental paradigm yields up to 100% transduction efficiency.Cells were then treated with MG-132 for 48 h and viability wasdetermined using Cell Titer 96 Aqueous assay (Promega).

(c) a-synuclein model. Mouse a-synuclein coding region was PCR-amplifiedfrom a mouse brain cDNA and fused at the C-terminus with a redfluorescent protein (RFP) dsRed2 to generate pa-syn-RFP. This plasmidwas co-transfected with pAAV.GFP or pAAV.dXIAP into SH-SY5Y cells andapoptotic cells were scored in 24 h using YO-PRO-1 dye (MolecularProbes). All transfections were performed using FuGene6 (Roche).

(iv) Stereotaxic Surgery and an In Vivo Model of Parkinson's Disease

Male rats (250-300 g) were used in this study. After a rat was placed ina stereotaxic frame, 2 μl of each vector (2×10⁹ genomic particles) inPBS was injected into the substantia nigra pars compacta over 10 minusing a 10-ml syringe and an infusion pump (World PrecisionInstruments). Animals received injections of AAV.GFP and AAV.dXIAP oneither side. Three months after surgery, rats were treated with DMSO(control) or a proteasome inhibitor PSI (3 mg/kg, i.p., every other day,eight injections total). The rats were sacrificed four weeks after thelast PSI injection. The animals were perfused with 4% paraformaldehydeand brains were analyzed by immunocytochemistry using a free floatingsections method.

Example 2 XIAP Protects SH-SY5Y Cells from 6-OHDA-Induced Apoptosis

This example describes how an inhibitor of apoptosis protein, XIAP,protects from 6-OHDA-induced apoptosis. SH-SY5Y cells were transfectedwith indicated plasmids, treated with 70 nM 6-OHDA and stained withpropidium iodide. The results from the study are shown in FIG. 2A. Notea significant reduction of cell death by either XIAP or dXIAP comparedto a control. All transfections were performed in triplicates. *p<0.001.

The location of apoptotic proteins was determined in an apoptosis assayusing the pCaspase3-Sensor vector shown in FIG. 2B. The caspase-3/7cleavage site appears in the DEVD region of the vector. The vector wastransfected into cells and the results are shown in FIG. 2C. This fusionprotein resides predominantly in the cytoplasm in normal cells (green).During apoptosis a nuclear exclusion signal (NES) at the N-terminus iscleaved by caspase-3 and the protein quickly translocates into thenucleus due to the presence of the nuclear localization signal (NLS) atthe C-terminus.

To determine the effect on caspase-3 activation, SH-SY5Y cells wereco-transfected with indicated plasmids and pCaspase3-Sensor vector for48 h, treated with 50 nM 6-OHDA, and scored using propidium iodidestaining in 6 h. Both XIAP and dXIAP significantly inhibited caspase-3activation as shown in FIG. 2D. All transfections were performed intriplicates. *p<0.001.

Example 3 XIAP is Protective in In Vitro Models of Familial Parkinson'sand Huntington's Diseases

This example describes how an inhibitor of apoptosis protein, XIAP, isprotective in in vitro models of familial Parkinson's and Huntington'sdiseases. pAAV.GFP and pAAV.dXIAP were cotransfected into SH-SY5Y cellswith pα-syn-RFP (FIG. 3A) or pQ111-RFP encoding for RFP fusions of themurine α-synuclein and the first exon of the mouse Huntington genecontaining additional polyglutamine repeats, respectively. Apoptoticcells were scored 24 h post-transfection using YO-PRO-1 vital DNA dye(FIG. 3B). Note that live cells shown in the top panel (dashed arrow)are impermeable to YO-PRO-1 while cells undergoing apoptosis (solidarrow) are stained positive. *p<0.001. Phase contrast is shown in FIG.3C. The percentage of cells undergoing apoptosis is shown in FIG. 3D.

These results demonstrate that dXIAP is a potent inhibitor of apoptosisin several in vitro models of Parkinson's disease including 6-OHDAmodel, α-synuclein model as well as a proteasome inhibitor model. Inaddition, XIAP is protective in an in vitro model of Huntington'sdisease.

Example 4 rAAV-Mediated dXIAP Delivery Protects Against Cell DeathInduced by Inhibition of the Proteasome Pathway

To test the ability of an inhibitor of apoptosis protein protectingagainst cell death induced by inhibition of a proteasome pathway, U87-MGcells were infected with AAV.GFP or AAV.dXIAP for 48 h and treated withproteasome inhibitors MG-132 or PSI for additional 48 h. Staurosporine,a compound with a well-characterized apoptotic effect, was included as apositive control. Cell survival is depicted in FIGS. 4A-4C. Theexpression of recombinant proteins is shown in FIG. 4D. *p<0.001.

Example 5 XIAP Protects Nigral Neurons in a PSI Model of Parkinson'sDisease

To determine whether an inhibitor of apoptosis protein protects neuroncells, rats were injected with AAV.GFP and AAV.dXIAP. These vectors wereinjected into the substantia nigra pars compacta (SNc) on each side andtreated with DMSO (vehicle) or PSI as described in Example 1(iv). Theresults from the study are shown in FIGS. 5A-5F, as wells as the bargraph in FIG. 5G. Note a significant reduction in the number ofTH-immunoreactive cells in SNc injected with the control virus followingPSI treatment while AAV.dXIAP virtually completely prevented cell loss.*p<0.001 as determined using ANOVA.

These results demonstrate that dXIAP delivered by a rAAV vector providesa long-term protection of dopaminergic neurons in a in vivo PSI model ofParkinson's disease. Slowly progressing nigral degeneration triggered byproteasome inhibition is believed to be a close recapitulations of theevents that mark sporadic Parkinson's disease in humans. This study isthe first demonstration that neuronal loss can be prevented in thismodel.

Example 6 Mutations of XIAP

To determine the effect of point mutations in XIAP peptide fragments,selected amino acid substitutions were made, including the following:D148A (Aspartate to Alanine); D214S (Aspartate to Serine); W310A(Tryptophan to Alanine); E314S (Glutamic Acid to Serine); and H343A(Histidine to Alanine) in dXIAP. The D148A and H343A substitutions weremade both singly and jointly. A XIAP mutant containing both the D214Sand E314S substitutions was made and tested. Finally, a mutantcontaining the D148A, D214S and W310A substitutions was made and tested.It should understood that the XIAP mutations of the invention includeany of the disclosed mutations either alone or in combination with otherdisclosed mutations.

FIG. 6A shows the known amino acid sequence of human XIAP (SEQ ID NO: 1)including the BIR1, Linker, BIR2, BIR3 and RING domains. FIG. 6B showsthe amino acid sequence of the BIR3 domain of human XIAP (SEQ ID NO: 2).The locations of the various mutations performed on XIAP are shown inFIG. 6C.

The mutant peptides were tested in the assays described above and theresults are shown in FIGS. 7-10. FIGS. 7 and 8 show that XIAP pointmutants that can no longer bind caspases are still protective inPSI-induced-, and polyglutamine induced cell death models, respectively.In contrast, when the ability of XIAP to bind and inactivate Smac isblocked (the last two mutants in FIGS. 7 and 8), XIAP is no longerprotective. Smac is a novel protein which promotes caspase activation inthe cytochrome c/Apaf-1/caspase-9 pathway. Smac promotes caspase-9activation by binding to inhibitor of apoptosis proteins, IAPB, andremoving their inhibitory activity. Smac is normally a mitochondrialprotein but is released into the cytosol when cells undergo apoptosis.Mitochondrial import and cleavage of its signal peptide are required forSmac to gain its apoptotic activity. These data suggest that the mainfunction of XIAP in these two models of neurodegenerative diseases is toblock Smac or its homologues with similar functions but not caspases.Additional data in FIGS. 9 and 10 show that synthetic inhibitors ofcaspases 3 and 9 alone, or in combination, cannot mimic XIAP effects.

The previous data demonstrates that the RING domain of XIAP appears tohave no significant effect in these studies and, as such, is notnecessary for the purposes of neuroprotection. The data in this example,further demonstrates that the function of XIAP can be localized to asmall portion of XIAP (BIR3 domain). Based on this discovery, smallneuroprotective peptides comprising this domain can be generated. Thesepeptides can be modified by adding leader signal peptides and peptidesresulting in a chimeric peptide. For example, a signal peptide and a TATdomain can be attached to the XIAP peptide fragment to allow it besecreted and make it cell permeable. One example of a chimeric peptideconstruct is a signal peptide-BIR3-TAT construct. This chimeric proteincan then be expressed by a viral vector or can be produced in vitro andinjected systemically.

1. A method for treating a neurodegenerative disease in a subjectcomprising: identifying a target site in the central nervous system thatrequires modification; and delivering a composition comprising anucleotide sequence encoding X-linked inhibitor of apoptosis protein(XIAP), or a peptide fragment thereof, and a pharmaceutically acceptablecarrier, wherein the composition is delivered to the target site in thecentral nervous system to treat or reduce the neurodegenerative disease.2. The method of claim 1, wherein the XIAP fragment is a 449 amino acidprotein.
 3. The method of claim 1, wherein the neurodegenerative diseaseis associated with protein aggregates.
 4. The method of claim 3, whereinthe neurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease, seniledementia, and Amyloid Lateral Schlerosis (ALS). 5.-13. (canceled) 14.The method of claim 1, wherein the composition is delivered usingstereotaxic delivery.
 15. The method of claim 1, wherein the target sitein the central nervous system is a region of the brain.
 16. The methodof claim 15, wherein the region of the brain is selected from the groupconsisting of basal ganglia, subthalmic nucleus (STN), pedunculopontinenucleus (PPN), substantia nigra (SN), thalamus, hippocampus, cortex, andcombinations thereof.
 17. The method of claim 15, wherein the region ofbrain is the substantia nigra pars compacta. 18.-39. (canceled)
 40. Amethod for treating a neurodegenerative disease in a subject comprising:identifying a target site in the central nervous system that requiresmodification; delivering a nucleotide sequence encoding an amino acidfragment of X-linked inhibitor of apoptosis protein (XIAP) to the targetsite in the central nervous system; and expressing the XIAP in thetarget site to treat or reduce the neurodegenerative disease.
 41. Themethod of claim 40, wherein the XIAP fragment is a 449 amino acidprotein of SEQ ID No.
 8. 42. The method of claim 40, wherein theneurodegenerative disease is associated with protein aggregates.
 43. Themethod of claim 42, wherein the neurodegenerative disease is selectedfrom the group consisting of Parkinson's disease, Huntington's disease,Alzheimer's disease, senile dementia, and Amyloid Lateral Schlerosis(ALS). 44.-45. (canceled)
 46. The method of claim 40, wherein thenucleotide sequence is delivered using a liposome-mediated deliveryvector.
 47. The method of claim 40, wherein the nucleotide sequencevermis delivered using stereotaxic delivery.
 48. The method of claim 40,wherein the target site in the central nervous system is a region of thebrain.
 49. The method of claim 48, wherein the region of the brain isselected from the group consisting of basal ganglia, subthalmic nucleus(STN), pedunculopontine nucleus (PPN), substantia nigra (SN), thalamus,hippocampus, cortex, and combinations thereof.
 50. The method of claim48, wherein the region of brain is the substantia nigra pars compacta.51.-73. (canceled)
 74. The method of claim 1, wherein the XIAP fragmentis a 449 amino acid fragment consisting of the XIAP open reading framelacking 48 C-terminal amino acids and protects dopaminergic neurons. 75.The method of claim 40, wherein the XIAP fragment is a 449 amino acidprotein selected from the group consisting of SEQ ID No. 8, SEQ ID No.13, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, SEQ IDNo. 27, SEQ ID No. 30, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 36, SEQID No. 37, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43,SEQ ID No. 46, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 51, SEQ ID No.52, SEQ ID No. 53, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ IDNo. 58, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 63, SEQID No. 64, and SEQ ID No.
 65. 76. The method of claim 40, wherein theXIAP fragment is SEQ ID No. 8 variant consisting of a 449 amino acidprotein selected from the group consisting of SEQ ID No. 13, SEQ ID No.17, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 27, SEQ IDNo. 30, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 37, SEQID No. 38, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 46,SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No.53, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ IDNo. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 63, SEQ ID No. 64, andSEQ ID No.
 65. 77. The method of claim 40, wherein the XIAP fragment isa 449 amino acid fragment consisting of the XIAP open reading framelacking the 48 C-terminal amino acids and protects dopaminergic neurons.78. The method of claim 40, wherein the XIAP fragment is a 449 aminoacid fragment consisting of the XIAP open reading frame having at least95% identity to SEQ ID No. 8 and protects dopaminergic neurons.
 79. Amethod for protecting dopaminergic neurons from apoptosis in a subjectcomprising: identifying a target site in the central nervous system thatrequires modification; and delivering a composition comprising anucleotide sequence encoding X-linked inhibitor of apoptosis protein(XIAP), or a peptide fragment thereof; and a pharmaceutically acceptablecarrier, wherein the composition is delivered to the target site in thecentral nervous system to protect at least one dopaminergic neuron fromapoptosis.
 80. The method of claim 79, wherein the XIAP fragment is a449 amino acid protein of SEQ ID No.
 8. 81. The method of claim 79,wherein the XIAP fragment is a 449 amino acid fragment consisting of theXIAP open reading frame having at least 95% identity to SEQ ID No. 8 andprotects dopaminergic neurons.
 82. The method of claim 79, wherein theneurodegenerative disease is associated with protein aggregates.
 83. Themethod of claim 79, wherein the subject has a disorder selected from thegroup consisting of Parkinson's disease, Huntington's disease,Alzheimer's disease, senile dementia, and Amyloid Lateral Schlerosis(ALS).
 84. The method of claim 79, wherein the composition is deliveredusing stereotaxic delivery.
 85. The method of claim 79, wherein thetarget site in the central nervous system is a region of the brain. 86.The method of claim 85, wherein the region of the brain is selected fromthe group consisting of basal ganglia, subthalmic nucleus (STN),pedunculopontine nucleus (PPN), substantia nigra (SN), thalamus,hippocampus, cortex, and combinations thereof.
 87. The method of claim85, wherein the region of brain is the substantia nigra pars compacta.88. The method of claim 79, wherein the XIAP fragment is a 449 aminoacid protein selected from the group consisting of SEQ ID No. 8, SEQ IDNo. 13, SEQ ID No. 17, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, SEQID No. 27, SEQ ID No. 30, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 36,SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No.43, SEQ ID No. 46, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 51, SEQ IDNo. 52, SEQ ID No. 53, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQID No. 58, SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 63,SEQ ID No. 64, and SEQ ID No.
 65. 89. The method of claim 79, whereinthe XIAP fragment is SEQ ID No. 8 variant consisting of a 449 amino acidprotein selected from the group consisting of SEQ ID No. 13, SEQ ID No.17, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 27, SEQ IDNo. 30, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 37, SEQID No. 38, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 46,SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No.53, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ IDNo. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 63, SEQ ID No. 64, andSEQ ID No. 65.